1. Field of the Invention
The invention relates to modification of oligonucleotides for incorporation of single or multiple reporter groups. More particularly the invention relates to improved modified oligonucleotides which are functionally derivatized to increase sensitivity of detection. Such oligonucleotides are useful as probes for a variety of nucleic acid-based diagnostic and therapeutic applications based on their hybridization to specific complementary nucleic acid sequences.
2. Summary of the Related Art
The preparation and use of functionalized oligonucleotides for incorporation of reporter groups is known in the art.
Agrawal et al., Nucleic Acids Res. 14:6227-6245 (1986) discloses introduction of biotin and flourescent dyes at either the 5' or 3' end of oligonucleotide. See also Agrawal, Tetrahedron Lett. 30: 7025-7028 (1989).
Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); Agrawal et al., J. Cell Biol. 107:468 (1988); and Haralambidis et al., Nucleic Acids Res. 18:501-505 (1989) teach the introduction of fluorophores into oligonucleotides.
These methods suffer from the limited signal strength inherent in the presence of the single reporter molecule which is incorporated into each oligonucleotide.
Consequently, numerous investigators have attempted to develop methods which allow the introduction of multiple reporter groups into each oligonucleotide as a means of increasing sensitivity of procedures which use the oligonucleotide as probes.
Fidanza et al., J. Am. Chem. Soc. 111:9117-9119 (1989), teaches the incorporation of reporter groups at phosphorothioate linkages in the nascent oligonucleotide.
Agrawal et al., Tetrahedron Lett. 31:1543-1546 (1990), and Agrawal et al., Nucleic Acids Res. 18:5419-5423 (1990), disclose methods for labelling oligonucleotides based on incorporating primary amines at phosphodiester moieties as phosphoramidates.
Nelson et al., Nucleic Acids Res. 127:7179-7186 (1989), discloses multiple reporter group incorporation at 5' termini of oligonucleotide using phosphoramidite linkage to the oligonucleotide via N-Fmoc-O.sup.1 -DMT-O.sup.2 -cyanoethoxydiisopropylaminophosphinyl-3-amino-1,2-propanediol, a fixed-length linker.
Misiura et al., Nucleic Acids Res. 18:4345-4354 (1990), discloses a method for incorporating multiple reporter groups on oligonucleotides via phosphoramidite linkage using a three carbon glyceryl attachment backbone to which the reporter group is connected by an ether-linked aminopropyl group, i,e., another fixed-length spacer.
Haralambidis et al., Nucleic Acids Research 18: 501-505 (1990), teaches linkage of multiple reporter groups solely to 3' ends of oligonucleotides using polyamide moieties connected to the 3' end of the oligonucleotides and lysine residues connecting the reporter groups to the polyamide moleties.
Although these methods are useful, they have many limitations. Spacing of reporter groups is a critical factor for increasing sensitivity of detection. The methods known in the art do not provide for controlled variation of the spacing of reporter groups and therefore may be limited in maximizing sensitivity. In addition, the chemistry in the known phosphoramidite methods is rather complex. While the use of polyamide and amino acid attachment means is amenable to some variation in spacing and involves a somewhat simpler chemistry, these attachment reagents due to inappropriate spacing can cause quenching of signal, at least with fluorescent reporter groups, thereby decreasing sensitivity.
There is, therefore, a need for improved methods for incorporating multiple reporter groups into oligonucleotide. Preferred improved methods would utilize a simpler chemistry than existing methods and would allow for controlled, variable spacing of reporter groups without causing quenching of signal.